Troxerutin Preconditioning Modulates the Activity of TLR4/NF-κB Signaling Pathway and Mitochondrial ATP-Sensitive Potassium Channels in a Rat Model of Myocardial Ischemia/Reperfusion Injury

Purpose Given the rising prevalence of ischemic heart diseases (IHD), investigations for achieving effective treatments are required. The purpose of this study is further understanding of troxerutin's impact on inammatory response induced by myocardial ischemia/reperfusion (MI/R) injury, and the role of toll-like receptor 4 (TLR4)-nuclear factor kappa B (NF-κB) signaling pathway and mitochondrial ATP-sensitive potassium (mitoK ATP ) channels in this scenario. cardioprotective effects against MI/R damage, which are partly mediated through anti-inammatory effects and the activation of mitoK ATP channels. via decreasing the activity of TLR4/NF-κB signaling pathway. We also indicated that the opening of mitoK ATP channels play crucial role in the cardioprotective effects of troxerutin in MI/R injury condition. However, further attempts examine the effects of troxerutin on the other important pathways involved in heart protection under I/R injury.


Introduction
Cardiovascular diseases are the major cause of death worldwide, especially in industrialized societies. So protecting the heart against stressors and ischemic conditions is very importan [1]. Myocardial ischemia/reperfusion (MI/R) injury is the tissue injury resulted when blood supply restores to the tissue following ischemia. Despite the fact that early reperfusion of the myocardial ischemic region is essential for the survival of cardiomyocytes, but reperfusion itself leads to the destructive responses in circulating cells and cardiomyocytes, and a series of pathophysiological and biochemical changes referred as reperfusion injury [2,3]. It has been shown that MI/R injury leads to the contractile dysfunction, arrhythmias and increased infarct size by increasing cardiomyocytes apoptosis and necrosis [4].
The pathophysiology of MI/R injury is complex, and several mechanisms mediate the detrimental effects of I/R injury [5][6][7][8]. One of the main mechanisms that causes MI/R injury is the activation of in ammatory pathways [9]. The in ammatory immune response has been shown to begin with ischemia and continue for several hours throughout the reperfusion phase. Toll-like receptor 4 (TLR4) is involved in the detrimental in ammatory effects under MI/R injury and participates in the formation of active oxygen free radicals, as well as activation of many cytokines [4,10]. TLR4 can activate and transfer the nuclear factor-kappa B (NF-κB) to the nucleus, thereby activating the transcription and releasing of in ammatory cytokines. Activation of NF-κB leads to the promotion of in ammatory genes expression such as tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β). Accumulation of TNF-α and IL-1β in the ischemic region causes tissue injury, oxygen free radicals releasing, cardiomyocyte damage and cardiac dysfunction [10,11]. In ammation has recently been considered as a therapeutic target in myocardial I/R injury, but the results are controversial. Despite numerous studies, the application of strategies targeting the in ammatory cascades in patients with myocardial infarction has been failed [12]. Therefore, researches in this eld and nding better strategies to combat with MI/R injury via the interventions in in ammatory pathways are of particular importance. Additionally, numerous studies have shown that the opening of mitochondrial ATP-sensitive potassium (mitoK ATP ) channel in the outer membrane of the mitochondrion may play essential role in preventing cardiac I/R injury via maintaining mitochondrion integrity [13,14]. Opening of mitoK ATP channels has been shown to play a key role in the anti-apoptotic processes and cardioprotective mechanisms of ischemic preconditioning and postconditioning [15]. Therefore, investigations for nding effective strategies to target mitochondrial K ATP channels in order to alleviate MI/R injury are very necessary.
Troxerutin, also known as vitamin P4, is a natural bio avonoid widely exists in tea, coffee, cereal sprouts, and various fruits and vegetables. It is derived from Sophora Japonica and Dimorphandra Gardneriana, and has anti-oxidant, anti-in ammatory, and anti-thrombotic activities [16][17][18]. Troxerutin has been commonly used in the treatment of chronic venous insu ciency [19]. It has been shown that troxerutin can reduce renal damage induced by d-galactose in mice by increasing the anti-oxidant activity and H 2 O 2 cleavage, and reducing MDA production, as well as inhibition of NF-κB-mediated in ammatory response [16]. Also, troxerutin can attenuate reactive oxygen species (ROS) production and DNA oxidative injury in d-galactose-induced kidney injury in mice [20].
To our knowledge, a less promising study is existed to determine the cardioprotective activities of troxerutin against I/R injury, especially via modulation of the activity of TLR4/NF-κB signaling pathway and mitochondrial K ATP channel. Given the potential anti-in ammatory effect of troxerutin and the lack of a study in this area, we hypothesized that troxerutin might decrease cardiac I/R injury via affecting the TLR4/NF-κB signaling pathway and mitochondrial K ATP channels. For that reason, the aim of the present study is to investigate the effect of troxerutin on myocardial infarct size, lactate dehydrogenase (LDH) release, TLR4 and NF-κB expressions, as well as TNF-α and IL-1β levels, and the role of mitoK ATP channels under MI/R injury. Insights into these aspects are expected to contribute to a better understanding of troxerutin's effects on MI/R injury.

Animals and chemicals
Healthy male Wistar rats at 3-4 months of age (n = 60, 250-300 g) were purchased from the animal center of Tabriz University of Medical Sciences. All the rats were housed in the animal room under a 12 h light/12 h dark schedule at a temperature of 25 ± 2°C and a humidity of 50 ± 10%, and had free access to standard food and water. Study protocols and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8th Edition, 2.2. Langendorff isolated heart perfusion and induction of regional I/R protocol Animals were heparinized (500 IU/kg), then anesthetized with intraperitoneal injection of a combination of ketamine (60 mg/kg) and xylazine (5 mg/kg). After opening the chest in layers, the heart was rapidly removed and then mounted on a pressure-constant Langendorff perfusion apparatus (ML176-V; AD Instruments, New South Wales, Australia). The isolated heart was retrogradely perfused via the aorta with K-H solution that contained (in mmol/L) MgSO 4 1.2; KCl 4.8; NaCl 118.5; NaHCO 3 25; KH 2 PO 4 1.2; CaCl 2 1.7 and glucose 11.1 at pH 7.4 under a constant perfusion pressure of 80 mm Hg. The perfusion solution was gassed with a mixture of 95% O 2 and 5% CO 2 at 37 ± 0.5°C. Throughout the stabilization, the coronary ow rate was 12-14 ml/min. For maintaining the temperature near 37°C, the isolated heart was surrounded by a homoeothermic glass cover. Before the induction of regional ischemia, the isolated hearts in Langendorff setting were perfused with K-H solution for 15-20 minutes to stabilize cardiac activity. Then regional ischemia was done by ligation of the left anterior descending (LAD) coronary for 30 min, close to its origin. Reperfusion was done by reopening of the LAD for 45 min. The e ciency of coronary ligation and su cient perfusion were approved by coronary ow. Declining at least 25% in coronary ow during occlusion was considered satisfactory.
In TXR-receiving groups, troxerutin (150 mg/kg) was orally administrated through a gavage tube once a day for 4 weeks before the induction of MI/R. In 5-HD-receiving groups, the hearts were perfused with a K-H solution containing 100 µM 5-hydroxydecanoate (5-HD, as a mitoK ATP channel blocker) 20 min before the ischemia. After 45 min of reperfusion, biochemical evaluations were performed from tissue sampling (n = 5), and the infarct size (IS) were evaluated (n = 5).

Evaluation of area at risk and infarct size
In order to differentiate the ischemic and non-ischemic areas from each other, the LAD was reoccluded following 45 min of reperfusion, then 0.25% Evans blue dye (3-4 ml, Sigma) was infused via the aortic cannula into the coronary system. After the hearts were frozen at -20°C for 24 h, the hearts were sliced into 2 mm sections, then incubated for 15 min in 1% 2,3,5-triphenyltetrazolium chloride phosphatebuffered solution (pH 7.4) at 37°C. Hence, the slices were placed in 10% formalin for 24-48 h. By means of Image J software (NIH, Bethesda, USA), the determination of infarct size was performed. Accordingly, the area at risk (AAR) was reported as a percentage of left ventricle (AAR/LV × 100), and IS was reported as a percentage of AAR (IS/AAR × 100).

Measurement of lactate dehydrogenase (LDH) release
LDH release into the coronary e uent was measured by a colorimetric method using the speci c laboratory kit (Pars Azmoon Co., Karaj, Iran) and autoanalyzer (Alcyon 300, Abbott Labs, Santa Clara, CA, USA) in compliance with the manufacturer's methods. A spectrophotometer detected the absorbance of the solution for LDH at 492 nm [21]. The results are reported in IU/L.

Preparation of tissue homogenates
45 min after reperfusion, the heart samples isolated from the ischemic zone of left ventricle were immediately frozen in liquid nitrogen and stored at -80°C. The preparation of samples was in accordance to a modi ed protocol for tissue homogenization. The ventricular tissue was weighed, then cut into pieces in about 5 ml of ice-cold lysis buffer (1 mM EDTA, 1 mM KCL, 1mM Na 3 VO 4 , 1 mM KH 2 PO 4 , 50 mM Tris-HCl pH 7.4, 1 mM NaF, 1% triton X100 and protease inhibitor cocktail) and then homogenized by means of a Polytron PT-10/ST homogenizer. After centrifuging the homogenate (10,000 RCF, 10 min, 4°C), the supernatants were removed from the homogenates and rapidly frozen at -80°C. To determine protein concentration in samples, Bradford method was used.

Western blotting
TLR4 and NF-κB in isolated hearts of all groups were measured by western blotting. Fresh-frozen samples were dissected and homogenized in radio-immunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The resulting solutions were centrifuged at 13,000 g for 20 min. Next, the supernatants were collected. Using UV 3000 ultraviolet spectrophotometer (NanoDrop, Wilmington, DE), the concentration of total protein was determined. Then, equal amounts of proteins (50 µg) were loaded into the electrophoresis chamber in 10-15% SDS-polyacrylamide gel. After that, separated soluble proteins were electro-transferred to a polyvinylidene-di uoride membrane (PVDF, Sigma-Aldrich, St. Louis, MO, USA), then incubated with 5% non-fat dry milk solution in Tris buffered saline-Tween 20 (TBST, pH 7.4) in room temperature for 2 h to block non-speci c bindings. The membranes were incubated with primary antibodies at 1:1000 against TLR4 and NF-κB (Cell Signaling Technology, USA) diluted in blocking buffer overnight at 4°C on a shaker. To control for equal loading, membranes were also incubated for β-actin antibody (1:1000, Cell Signaling Technology, USA), diluted in TBST overnight at 4°C. Then blots were incubated with goat anti-rabbit horseradish peroxidase conjugated IgG for 1 h at room temperature. For detecting the immunoreactivity, protein bands were incubated with enhanced chemiluminescence (ECL) substrate (Millipore) and then were exposed to X-ray hyper lm inside a hypercassette in a darkroom for 5 min, and the chemiluminescence of the antibody binding was visualized using a visualizing machine. In order to quantify the intensity of protein bands in the blots, densitometry analysis was used and then normalized with the intensity of β-actin band as loading control. The measured values are reported in arbitrary unit.

Determination of in ammatory cytokines
For measuring the levels of in ammatory cytokines (TNF-α and IL-1β), rat-speci c enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, Bender Medsystems, Austria) were used according to the manufacturer's methods. First, frozen samples get warmer in room temperature, then mixed gently. 50 ml samples and standards were added in duplicate to the 96-well plate. After adding the polyclonal antibodies separately to all wells, their surface were covered and incubated for 2 h at room temperature. Following washing, streptavidin-HRP was added to all of the wells and were incubated for 1 h at room temperature. TMB substrate solution was added to all of the wells. In order to hamper the enzyme reaction, the stop solution was added into the each well. Using a spectrophotometer, the relative absorbance of cytokines was read at 450 nm. The resultant values were normalized and expressed as pg/mg of protein in each sample. The limits of detection of rat TNF-α and IL-1β with ELISA kits were determined to be 11 and 4 pg/ml up to 2500 pg/ml, respectively. The calculated overall intra-assay and inter-assay coe cients of variation for TNF-α were < 5 and < 10 % and for IL-1β were < 10 and < 10 %, respectively. According to the manufacturer's assays, these ELISA kits show no cross-reactivity with the other cytokines and molecules.

Statistical analysis
The analysis was carried out using SPSS software 25 (SPSS Inc., Chicago, IL, USA). In order to determine the statistical signi cances between groups, one-way analysis of variance (ANOVA) followed by Tukey post hoc test was used. All values are expressed as means ± standard errors of means (SEM). P value below 0.05 (P < 0.05) was considered as statistically signi cant difference.

Myocardial area at risk (AAR) and infarct size (IS)
As shown in Fig. 1, there were no signi cant differences in the area at risk between all groups. The IS was signi cantly increased in MI/R group as compared to Control group (P < 0.001). Preadministration of troxerutin signi cantly reduced IS as compared to MI/R hearts (P < 0.05). Adding 5-HD to the perfusion solution signi cantly abolished the IS lowering effect of troxerutin (P < 0.05). However, there was no signi cant change in IS between MI/R and MI/R + 5-HD groups (Fig. 1). Figure 2 shows the levels of LDH release into the coronary e uent of treated and untreated I/R hearts.

LDH release
The levels of LDH release were signi cantly increased in MI/R group as compared to Control group (P < 0.001). Preadministration of troxerutin signi cantly reduced the levels of LDH release into the coronary e uent during reperfusion as compared to MI/R hearts (P < 0.05). Adding 5-HD to the perfusion solution signi cantly abolished the LDH lowering effect of troxerutin (P < 0.05). No signi cant differences were found in LDH release between MI/R and MI/R + 5-HD groups (Fig. 2).

TLR4 expression
As shown in Fig. 3, the expression of TLR4 in MI/R group was signi cantly increased as compared to Control group (P < 0.01). Preadministration of troxerutin signi cantly reduced the expression of TLR4 as compared to MI/R hearts (P < 0.05). However, troxerutin in the presence of 5-HD could not signi cantly decrease the expression of TLR4 as compared to MI/R + TXR group. In other words, administration of 5-HD reversed the effect of troxerutin on the alteration of TLR4 expression. No signi cant differences were found in TLR4 expression between MI/R and MI/R + 5-HD groups (Fig. 3). Figure 4 shows the effect of troxerutin on the expression of NF-κB in treated and untreated I/R hearts. The expression of NF-κB in MI/R group was signi cantly increased as compared to Control group (P < 0.001). Preconditioning with troxerutin signi cantly reduced the expression of NF-κB as compared to MI/R hearts (P < 0.01). Blocking the mitoK ATP channels by 5-HD signi cantly abolished the NF-κB-lowering in uence of troxerutin (P < 0.05). No signi cant differences were found in NF-κB expression between MI/R and MI/R + 5-HD groups (Fig. 4). 3.5. The levels of TNF-α As shown in Fig. 5, the levels of TNF-α in MI/R group was signi cantly increased as compared to Control group (P < 0.001). Preadministration of troxerutin signi cantly reduced the levels of TNF-α as compared to MI/R hearts (P < 0.01). However, blocking the mitoK ATP channels by 5-HD signi cantly abolished the TNF-α-lowering effect of troxerutin (P < 0.05). No signi cant differences were found in TNF-α levels between MI/R and MI/R + 5-HD groups (Fig. 5).

NF-κB expression
3.6. The levels of IL-1β Figure 6 shows the effect of troxerutin on the levels of IL-1β in treated and untreated I/R hearts. The levels of IL-1β in MI/R group was signi cantly increased as compared to Control group (P < 0.001). Preconditioning with troxerutin signi cantly reduced the levels of IL-1β as compared to MI/R hearts (P < 0.05). In addition, the levels of IL-1β were signi cantly decreased in troxerutin-receiving group without experiencing I/R as compared to Control group (P < 0.05). Blocking the mitoK ATP channels by 5-HD signi cantly abolished the IL-1β-lowering in uence of troxerutin (P < 0.05). No signi cant differences were found in the levels of IL-1β between MI/R and MI/R + 5-HD groups (Fig. 6).

Discussion
The present study assessed the therapeutic potentials of troxerutin in mitigation of MI/R damage in isolated rat heart. By evaluating clinically relevant end points, this study proved that preadministration of troxerutin is able to reduce cardiac infarct size and LDH release following MI/R compared with the untreated I/R myocardium. Besides, this study documented that the positive effects of troxerutin preconditioning may be mediated in part by its effect on in ammation. In more details, troxerutin preconditioning showed potent anti-in ammatory and cardioprotective activity via decreasing TNF-α and IL-1β levels. Also, troxerutin was found to suppress in ammatory response by reducing the activity of TLR4/NF-κB signaling pathway. In addition, our results showed that the bene cial effect of troxerutin was reversed by using 5-HD, as a mitoK ATP channel blocker. So it can be said that troxorutein may exert its cardioprotective effect under MI/R injury by the opening of mitoK ATP channels.
In previous experimental studies of MI/R models, the bene cial properties of troxerutin have been evaluated. However, in order to achieve clinical goals in future, we need to widely study the effects of troxerutin on cellular signalings. For that reason, several experimental studies have assessed cardioprotective effects of troxerutin by evaluation of important cellular signalings, but the mechanism of troxerutin on in ammation via TLR4/NF-κB signaling pathway in MI/R damage has never been studied in depth. It seems that speci c modulation of TLR4/NF-κB signaling pathway by troxerutin might be a novel strategy to increase survival under MI/R injury in the future. It should be noted that the results obtained from the present study can lead to the better understanding of cardioprotective activity of troxerutin as a natural bio avonoid in MI/R injury. The most signi cant observation of the current work is that troxerutin preconditioning reduced I/R injury, which was con rmed by reduction of cardiac infarct size and LDH levels. It means that pretreatment with troxerutin can increase cardiac resistance to I/R injury. Our work demonstrated that cardioprotective effects of troxerutin in I/R injury were accompanied by its bene cial effects on in ammation through reduction of in ammatory cytokines such as TNF-α and IL-1β levels, as well as reduction of TLR4 and NF-κB expressions.
The speci c pathophysiological mechanisms underlying MI/R injury are complex and needs further investigations; however, several studies demonstrated that in ammatory response takes part in MI/R injury. It has been proven that an important protein which is involved in the in ammatory response of MI/R damage is TLR4 [10]. In particular, TLR4 triggers the cytokines releasing, neutrophil activation, and elevation in ROS following MI/R injury [22][23][24]. TLR4 plays its role via MyD88 pathway, and NF-κB can be activated through this dependant pathway, so the associated in ammatory cytokines will be released. It has been proven that the NF-κB pathway can be activated under myocardial ischemia, and can be further activated under reperfusion. So it has a decisive role in MI/R injury and participates in the stress reaction and tissue damage [10,25]. After translocation of NF-κB to the nucleus within the rst few minutes following reperfusion, NF-κB promotes cell dysfunction and death by induction of pro-in ammatory and pro-apoptotic genes expression [4]. Therefore, inhibition of TLR4/NF-κB signaling pathway can be considered as an important therapeutic target for attenuation of MI/R injury. In consistent with previous studies, our data showed that I/R led to the increase in TLR4 and NF-κB expressions, and this accelerated in ammatory response causes a decrease in cardiac resistance to reperfusion injury. Interestingly, preadministration of troxerutin for 4 weeks could alleviate myocardial I/R-induced elevation of TLR4 and NF-κB expressions, and ultimately protect the heart against I/R damage. So it can be said that troxerutin has potent anti-in ammatory property via reducing the activity of TLR4-NF-κB pathway.
TNF-α and IL-1β play the key role in MI/R injury via causing cardiac dysfunction and cardiomyocyte necrosis and apoptosis [10]. It has been demonstrated that activation of myocardial TNF-α and TNF receptor have complex role in MI/R damage and protection from it. Excessive expression of TNF-α and stimulation of cardiomyocyte TNF receptor type 1 can lead to cardiac cell death, brosis, hypertrophy and contractile dysfunction. Whereas, lower expression of TNF-α and stimulation of cardiomyocyte TNF receptor type 2 are protective [26]. Our study revealed that I/R caused a signi cant increase in the myocardial levels of TNF-α and IL-1β, which was associated with the increased cardiac injury in comparison with healthy hearts. However, pretreatment with troxerutin caused decreased in the myocardial levels of TNF-α and IL-1β. Thus, positive effect of troxerutin on myocardial I/R damage may be attributed to the reduction of in ammatory cytokines such as TNF-α and IL-1β via decreasing the activity of TLR4-NF-κB signaling pathway.
Studies have demonstrated that mitoK ATP channel, which is considered as end effector of cardioprotective pathways, has cardioprotective activity in MI/R injury by regulation of the mitochondrial matrix volume, and decreasing in both calcium uptake by mitochondria and mitochondrial membrane potential [27]. It has been suggested that the mitoK ATP channel opening decreases pro-apoptotic mediators and improves mitochondrial function [15], while administration of 5-HD, as a mitoK ATP channel blocker, reverses the reducing effect of preconditioning and postconditioning on infarct size [28]. In the present study, we showed that the anti-in ammatory effects of troxerutin were reversed when the hearts were perfused with a K-H solution containing 5-HD, 20 min before the ischemia. So it can be said that the positive effects of troxerutin may partly be mediated through the mitoK ATP channels opening, and probably there is an association between the activation of mitoK ATP channels and inhibitory effect of troxerutin on in ammatory mediators under MI/R injury, but needs to be investigated in depth. In addition, it seems that the activation of mitoK ATP channels by troxerutin can protect the functional integrity of mitochondria, prevent the mitochondria swelling, and inhibit the mitochondrial permeability transition pore (mPTP) opening, leading to the decreased production and releasing of in ammatory mediators from the mitochondria in MI/R condition.
Conforming to our study, many previous researches demonstrated that troxerutin has protective effects against cardiovascular diseases, and oxidative and in ammatory damages in numerous organs [16-19, 29, 30]. Troxerutin has been shown to inhibit the endoplasmic reticulum stress pathway and reduce intracellular in ammatory proteins and enzyme activities including COX2, JNK1/IKKb, iNOS, and NF-κB in speci c tissues of healthy rats [20,[30][31][32]. Moreover, in our previous works, we have indicated that troxerutin preconditioning could protect the diabetic I/R hearts by prevention of myocardial apoptosis through glycogen synthase kinase-3β (GSK-3β) phosphorylation [33]. In addition, we proved that troxerutin preconditioning had protective effects in doxorubicin-induced cardiotoxicity in rats via antioxidative activities and restoration of mitochondrial function and the expression pro les of cardiac SIRT-1/PGC-1α/NRF-2 network [34].

Conclusions
The results of the present work contribute to some valuable conclusions. Preconditioning with troxerutin could provide cardioprotective effects under MI/R damage which were proven by reduction of myocardial infarct size and LDH release. Cardioprotective effects of troxerutin were partly mediated through modulation of in ammatory response including reduction of TNF-α and IL-1β via decreasing the activity of TLR4/NF-κB signaling pathway. We also indicated that the opening of mitoK ATP channels play a crucial role in the cardioprotective effects of troxerutin in MI/R injury condition. However, further attempts are required to examine the effects of troxerutin on the other important pathways involved in heart protection under I/R injury.

Funding
The author(s) disclosed receipt of the following nancial support for the research, authorship, and/or publication of this article: This work has been supported by a grant (grant number: 60872) from Drug Applied Research Center, and Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Islamic Republic of Iran.

Con icts of interest
The author(s) declared no potential con icts of interest with respect to the research, authorship, and/or publication of this article.

Availability of data and material
The authors con rm that the data supporting the ndings of this study are available within the article [and/or] its supplementary materials.
Code availability Not applicable.

Figure 3
Western blot band analysis and densitometry of TLR4 correlated to the β-actin band in different groups.